4
Ligand Screens What behaviors lie within the black box? What are its rules? Challenge: acquire, organize and interpret a data set that will allow us to  choose the cellular signaling probes that will be most informative  assemble and evaluate input/output data  constrain models of how the signaling pathways work in cells according to overall cellular behaviors The ligand screen is our first real experiment single- ligand screen is...

5
Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized?

6
Single Ligand Screen How many inputs are there ? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? How many regulatory ligands does a cell respond to ? How many do we have to look at ? What is the most efficient way to look ? Depends on # of signaling patterns

7
Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? What ligands are redundant? What ligands give overlapping outputs? How many ligands with “identical” outputs should we consider? How do we identify “identical” experimentally? Can response patterns be productively clusterd ?

8
Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? Can we identify conserved patterns of output patterns even if individual outputs differ? How do we look for them experimentally? Do different response patterns draw from a listable toolbox of signal outputs? Do different response patterns reflect branched pathways, mulitple receptors,...?

9
Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? Distinctive groupings of individual responses? Relative dynamics and time courses?

10
Single Ligand Screen How many inputs are there? How many patterns of response are there? What are the structures of these patterns? How do they differ? How are these patterns organized? Are there rules for network organization? What are the rules? How does a cell assemble its signaling network? Buzzwords: module, heirarchy, parallel How do you rigorously identify such patterns?

11
Double Ligand Screen R A+B = R A + R B ? Conceptual violations of signal additivity 0+0=1 1+1=0 1+0=0 1+1=17 1+0=17 How often do two signaling pathways interact to produce a response other than the sum of the two individual responses This screen is designed to tell us

12
Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? How do you identify such interactions conceptually? How do you identify them experimentally? What are the mechanisms of these interactions? Are the interactions regulated? (Triple-ligand screen...) Are there rules for network organization? What are they? R A+B = R A + R B ?

13
Double Ligand Screen How numerous (dense) are ligand - ligand interactions? R A+B = R A + R B ? How many ligand pairs must we consider? How complex a network should we plan on studying? How do we prepare our models to direct our experimental design? to map such a system? Does a cell process information more through complex interactions than with multiple signals ?

14
Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? R A+B = R A + R B ? Will receptors with similar individual outputs demonstrate similar spectra of double-ligand interactions? How does this result impact on our experiments?

16
Double Ligand Screen How numerous (dense) are ligand - ligand interactions? Which ligands interact? Are interactions determined primarily by individual outputs? How do you identify such interactions conceptually? How do you identify them experimentally? What are the mechanisms of these interactions? Are the interactions regulated? (Triple-ligand screen...) Are there rules for network organization? What are they? R A+B = R A + R B ?

17
established strategies for the single ligand screen. set up some of the wide array of necessary assays. executed a single-ligand screen in B cells and WEHI. begun to analyze single-ligand data. provocative enough that it won’t be “done” soon established strategies for the double-ligand screen. non-trivial and still under development time and concentration domains different kinds of interactions choice of measured outputs choices influenced by types of assays run partial double-ligand screen on B cells. How do we best and most efficiently execute and analyze the single- and double-ligand screen on a new cell type? So far, we have...